We are currently involved in a multidisciplinary collaboration to develop potent and specific inhibitors of human Chk2 kinase. Our role in this project is to determine co-crystal structures of Chk2 in complex with small molecule inhibitors. Using a library of over 100,000 compounds from the Open Repository Library to screen for novel inhibitors of Chk2, the Screening Technologies Branch identified a bis-guanylhydrazone, NSC 109555 (4,4-diacetyldiphenylurea-bis(guanylhydrazone), as a lead compound with an IC50 of 240 nM for Chk2. Biochemical characterization of this compound confirmed that it is a reversible and competitive inhibitor that targets the ATP binding pocket of Chk2. Initial kinase profiling of NSC 109555 against a panel of over 20 kinases demonstrated high selectivity of NSC 109555 for Chk2. To illuminate the molecular basis of Chk2 inhibition by NSC 109555 and other similar compounds, we initiated an effort to co-crystallize the enzyme with these small molecules. To this end, the catalytic domain of Chk2 was cloned, expressed as a His6-MBP fusion protein and purified to homogeneity in-house. We succeeded in determining the co-crystal structure of NSC 109555 in complex with the catalytic domain of Chk2 at 2.07 resolution by molecular replacement. The crystal structure confirmed that the inhibitor binds to the ATP-binding pocket of Chk2 in an elongated fashion, but, importantly, the mode of binding was different that what had been predicted by molecular modeling. NSC10955 is anchored to the active site via hydrogen bonding of one terminal guanylhydrazone moiety to Glu273 and water mediated hydrogen bonds between a backbone carbonyl of the inhibitor with Glu302 and Met304. A series of NSC 109555 analogs were subsequently synthesized by Provid Pharmaceuticals that included four different classes of structural modifications. The goals of the modifications were to achieve desymmetrization of the compound and scan a variety of substituents on the guanylhydrazone, alkyl, and aryl moieties. We have been able to determine co-crystal structures for at least one compound from each structural class, the most potent of which was PV1019 (IC50 = 15 nM, 2.07 ). Our success in determining the co-crystal structure of Chk2 in complex with PV1019 presented an opportunity for further structure-based optimization of the compound. PV1019 binds to the active site of Chk2 via direct hydrogen bonds of the guanylhydrazone moiety with Glu273 and the 2-nitro-indole with Glu302 and Met304. Our electron density maps also revealed the key roles of several well-ordered water molecules in the active site that form water-mediated hydrogen bonds between the carbonyl and amide backbone of PV1019 and Glu308 and Glu302. A noteworthy feature of the co-crystal structure is the presence of a hydrophobic cavity directly above the methyl functional group of PV1019 which differs slightly from the related Chk1 kinase. In Chk2, this cavity is composed entirely of hydrophobic residues, whereas in Chk1 there is one polar, hydrophilic residue, Asn59 (Leu277 in Chk2). We proposed that it might be possible to improve the specificity of the Chk2 inhibitor by optimizing the binding to this cavity. Current efforts are now focused on synthesizing analogs based on functional group substitutions on the neighboring methyl group next to the guanylhydrazone moiety which projects towards this cavity with the goal of optimizing the interactions between the inhibitor and this cavity. Provid has synthesized three new analogs based on structural characterization of the binding mode of PV1019 and we have successfully co-crystallized two of these compounds with Chk2;PV1322 (IC50 = 370 nM, 1.90 ) and PV1162 (IC50 = 12 nM, 2.2 ). A third analogue, PV1352, is especially promising, as is it has been shown to be approximately 5-fold more potent than PV1019. We have recently received this compound and are attempting to co-crystallize it with Chk2. The determination of the co-crystal structure of Chk2 in complex with PV1162 has also provided new details. PV1162 was created by altering the 2-nitro-indole group of PV1019 to a 5-methoxy-indole and an isopropyl moiety was attached to the methyl group of PV1019 in an effort to fill the hydrophobic cavity. The co-crystal of Chk2 with PV1162 reveals that the indole ring has now flipped over, thereby improving the hydrogen bonding network of the indole NH group with the active site. Additionally, the isopropyl group that was added to the methyl group now fits snuggly into the hydrophobic cavity. This compound will serve as a guide for the development of new analogs in which we examine additional substituents on the indole ring as well as additional hydrophobic substituents on the methyl group.